Method and apparatus for analyzing proteins

ABSTRACT

A method and an apparatus for determining the sequence of the amino acids in a protein. The protein is applied to the surface of a solid support arranged in a reactor. A vaporized coupling agent is supplied to the protein thus forming a protein derivative. The protein derivative is split at the first peptide binding by a supplied vaporized splitting agent and the amino acid derivative split off is transferred to an absorber. By raising the temperature of the absorber the amino acid derivative is then transferred to an analyzer, e.g., a gas chromatograph.

United States Patent Sjoquist 51 Feb. 29, 1972 [54] METHOD AND APPARATUSFOR 3,505,021 4/1970 Eveleigh ..23/230 ANALYZING PROTEINS 3,537,82111/1970 Hrdina ..23/230 [72] Inventor: John A. Sjoquist, Uppsala, Swedenprimary Examine, MorriS w 73 A LKBJ) duk' AB B S d Assistant ExaminerR.E. SCI'WiIl 1 Sslgnee m er romma we en Att0rneyChristen & Sabol [22]Filed: Apr. 9, 1970 21 Appl. No.: 26,977 [57] ABSTRACT A method and anapparatus for determining the sequence of [52] U s 23/230 R 23/230 M23/232 R the amino acids in a protein. The protein is applied to thesur- 23/232 23/253 23/254 face of a solid support arranged in a reactor.A vaporized [51] Int. Cl ..G01n31/06 00in 31/08 G Oln 33/00 couplingagent is supp'ied 9 [58] Field of Search 23/230 232 254 23 proteinderivative. The protein derivative is split at the first l peptidebinding by a supplied vaporized splitting agent and the amino acidderivative split off is transferred to an absorber. By 56] ReferencesCited raising the temperature of the absorber the amino acid deriva tiveis then transferred to an analyzer, e.g., a gas chromato- UNITED STATESPATENTS p 3,298,786 1/1967 Hinsvark ..23/230 16 Claims, 2 DrawingFigures METHOD AND APPARATUS FOR ANALYZING PROTEINS The presentinvention refers to a method and apparatus for determining the aminoacids in a protein. It is known per se to carry out such at determiningby making the protein react with a chemical compound, a so-calledcoupling agent, which is combined with the amino end of the proteinmolecule. The protein molecule is then split at the first peptinebinding. Thus, a remaining part of the protein and an amino acidderivative is formed. The amino acid or the amino acid derivative couldthen be identified for instance by means of gas chromatography. Thisprocedure is then repeated, i.e., the next amino acid is split from theprotein molecule, etc.

lt is known per se to carry out such a sequence determining in asolution. This procedure will, however, imply certain drawbacks, whichdrawbacks among other things are due to the fact that the solubility ofthe remaining part of the protein increases as the chain length of theprotein molecule is decreased. Furthermore, the procedure is rathercomplicated and lengthy. Another drawback consists therein that unwantedside reactions appear.

The above-mentioned drawbacks are reduced or eliminated by using thepresent invention according to which the analysis is carried out in gasphase and according to which the analysis is carried out in has phaseand according to which those groups in the protein molecule which giverise to said reactions are blocked. The method according to theinvention is characterized in, that the protein is applied on a solidsupport, that a coupling agent in gas phase is supplied to the protein,and that after the splitting the formed amino acid derivative is removedin gas phase. According to a preferred embodiment of the invention, theprotein applied on the support is treated with a blocking agent by meansof which reactive groups in the protein molecule are blocked and thusthe risk of obtaining unwanted side reactions of the protein and thecoupling agent is eliminated.

By using the method according to the invention a high degree of specificsplitting is obtained, i.e., only one amino acid is split from theprotein in each operation. Furthermore, very few unwanted side reactionsappear. According to the invention it is furthermore possible to use avery small quantity of protein, i.e., l nanomol or less is required. Asthe analysis is carried out in gas phase a fast determining is obtainedand furthermore an automatic control of the operation could easily beobtained.

The solid support of the protein is preferably a material that has acertain adsorbing effect on protein molecules but which has a very lowadsorbing effect on the split amino acid derivatives which have a smallmolecular mass. Glass has been found to have these properties. The glassis preferably etched, for instance with fluoric acid so as to obtain alarge surface. The glass can be used in the form of small beads,preferably having a diameter of less than 0.1 mm. so as to make ananalysis in a fluidized bed possible. Alternatively the glass could bein the form of a capillary, preferably having an inner diameter of O.3lmm. if the support is a powder the protein is preferably supplied as asolvent. The amount ofsolvent should be small in comparison with thequantity of powder so that the powder can adsorb the solvent withoutlosing its character of a dry powder. If the carrier has the form of acapillary, the protein solvent is pressed through the capillary tube andpart of the protein will be adsorbed on the wall of the capillary. ifthe support consists of powder the beads should be massive withoutmicropores as such pores would too much adsorb the compounds havingsmall mass numbers.

The coupling agent and other used reagents are supplied to the proteinapplied on the support by means of a carrier gas which should be freefrom oxygen and other impurities and for instance consist of nitrogen ora noble gas. The mixing of reagent and carrier has preferably takesplace in such a way that the carrier gas is introduced into a containerwhich contains the reagent. By regulating, e.g., the temperature of thecontainer and the flow velocity of the carrier gas the partial pressureof the reagent in the carrier gas can be regulated.

As a coupling agent an isothiocyanate is preferably used as thiscompound has a sufficient volatility to be supplied to the protein ingas phase. Suitable compounds are, e.g., tert butylisothiocyanate,allylisothiocyanate, methylisothiocyanate ortrimethylsilylisothiocyanate. The coupling agent should be mixed with avolatilizable buffer, e.g., trimethylamine, triethylamine or pyridineand water. The buffer is preferably mixed with a carrier gas asdescribed above, the buffer then being supplied to the carrier gasstream which contains the coupling agent. In order to make sure that aneffective mixing is obtained the gas mixture is preferably suppliedthrough a relatively narrow tube in which a turbulent flow could beobtained.

The reaction is preferably carried out in several steps, the first stepimplying a reaction between the coupling agent and the amino end of theprotein molecule and the second step implying a splitting of the proteinderivative by means of a supplied unhydric acid, e.g., formic acid,acetic acid, trifluoracetic acid, hydrogen chloride or compounds ofthese acids. When using a methylisothiocyanate as a coupling agent thefollowing reaction will take place:

The obtained prot einm will then be split as will be described below,the splitting implying a forming of a protein rest and a cyclicthiazolinone derivative of an amino acid:

H CHa-NZC-CHRr-CO CHa-NC: S 'NH'OHRICO Other coupling agents that can beused are alkalicyanates and -isothiocyanates, e.g.:

HN-CO-NH-CHRr-CO Dithiouretanes can also be used as coupling agents,e.g., ethyl-N-bensoyldithiocarbamate as described by the followingformula:

The compound (I) is split at the first peptide binding and istransformed into a stable thiohydantoine derivative according to thefollowing formula:

thiophosgene in combination with an amine, e.g.:

CHg-NCS-NH-CHRrCO The blocking agent should be coupled to reactivefunctional groups in the protein molecule so as to avoid that thecoupling agent or other reagents used react with these groups in anuncontrollable way. Thus, free carboxylic groups can be esterified by analcohol as methanol by using HCL, BF 'or BCL as a catalyzer or by usingdiazomethane, CH N The blocking of OH-NH or SH-groups can be carried outby acylation by an acetic acid anhydride, trifluoroucetic acidanhydride. pentafluoropropionic acid anhydride or h'ep tafluorobutyricacid anhydride. A selective blocking of NH5- groups can be carried outwithout using the above mentioned alkylisocyanates, oralkylisothiocyanates by using chloroformates. If, however, all thereactive groups in the protein molecule are to be blocked by using theabove mentioned blocking agents, it is necessary to carry out a seriesof successive reactions, i.e., one reaction for each kind of reactivegroup.

It is, however, possible to block all reactive groups in one singlereaction by using certain silicon containing blocking agents. Examplesof such a blocking agent are N- O,di(trimethylsilyl)acetamide:

O -Si(CHa)a CHa-C N-Si(CHa)a andN-O,di(trimethylsilyl)trifluoroacetamide which has the same formula butin'which the hydrogen of the methyl group is substituted by fluorine.Another coupling agent that can be used is hexamethyldisilazane, (CH3)3Si NH Si(CH The above-mentioned silicon compounds could also be used foranother purpose, namely to increase the volatility of the amino acidderivative split from the protein. If such an increase is performed theamino acid derivative could more easily be removed from the remainingpartof the protein by raising the temperature or lowering the pressureor both. Other agents, e.g., trimethylchlorosilane,imethyldichlorosilane, N-trimethylsilyldiethylamine, N-O,di-(dimethylsilyl)acetamide, N-trimethylsilyl-imidazole, might for instancebe used for increasing the volatility of the amino acid derivative.

The use of vapor pressure increasing agents also implies that the aminoacid derivative will be more resistant to variations in temperature.Thus, the amino acid derivative can be separated from the remaining partof the protein at a higher temperature.

Both these above-mentioned characteristics of the silicon compounds willbe illustrated by the following example.

A. A coupling agent consisting of methylisothiocyanate reacts with aprotein in which the first two amino acids are treonine and asparaginicacid:

011011 0112 R C tHa" H CHg-NH-CS-NH-CH-CO-NH-OH-CO-NH-CH-CO CHOH OH; R

B. The polar groups of the protein derivatives (l) are then made toreact with hexamethyldisilazane, thus blocking these polar groups sothat no uncontrolled side reactionscan appear: I 5

I (CHQa-Sl-NH-SKCHQE CHaNH-CS-NH'fiJH'C O -NH-CH-C O -NH-OH-C OOSi(CTT3)3 OH H; R $11 COO'SKCHa):

K ah (II) 15 O. The Corn ound II i th 1 t adding rmuomgcem Jag: s an spiat the first peptide binding, by

C Fz-C O OH CILPN'CB'NH'CH'CO CHOH CH; (III)HzN-(IJHCO-NH-(IDHCOOSi(CHa)a CH R COOSKCH (IV) 2CF -COOS1(CH D. Inorder to increase the volatility of the thiohydantoine (III), a siliconcompound is supplied, e.g., N-O,di(trimethylsilyl)acetamide, whichreacts with the thiohydantoine (III) as well as with the remaining partof the. portion (IV) according to the following formula: I

d-SKCHa): III -I- IV CH-C\ /Sl(CHa)a CHaeN-CB-N CH CO 1(CHa):(|3HOS1(CH;);

M CH: sh-Sl-NHCH'CO-NH-PHCO0Si(CHa)s 5 0 (III R ooosuom):

E. The compound Vnow hasa high vapor pressure and can easily be.separated from the compound V! by heating or evacuation or both. Thevaporized compound V could then be identified by methods known per se. v

F. Before the next step of the sequency procedure, the trimethylsilylegroups haveto be removed from the compound VI, especially andtrimethylsilyle group which is bound to the amino end of the compoundVI, as otherwise this group would prevent the reaction of the couplingagent. This procedure is carried out ,by supplying vaporized water,preferably in combination with an alcalic buffer, e.g., trimethylamine.The following hydrolysis then takes place:

H20 VI HzN-|CH-CO-NH-CH2COOH COOH The remaining part of the protein VIIis now in such a state that a new series of the steps A-F describedabove can be carried out.

The invention will now be described in detail with reference to theaccompanying drawing in which FIG. 1 shows an apparatus according to theinvention and FIG. 2 shows a part of the apparatus according to FIG. 1,namely the dosing apparatus in which the mixing of coupling agent andblocking agent with the carrier gas flow is carried out.

The apparatus shown in FIG. 1 mainly comprises a reactor 1 in which theprotein reacts, a dosing apparatus 2 in which the reagents used in theprecess are mixed with the carrier gas 3, an absorber 3 in which theamino acid derivatives are absorbed and an analyzer 6 in which the aminoacids are identified. The analyzer, which might consist of a gaschromatograph or some other apparatus known per se, does not form partof the invention and will thus not be described in detail.

The reactor 1 consists of a vertical tube, comprising a perforated plate11 on which a granulated solid support is applied. Gas can be suppliedvia a tube connected to the bottom of the reactor and the gas is drainedvia a tube connected to the top of the reactor. At a suitable gasvelocity the bed is fluidized. The reactor is provided with a scalableopening 13 through which a solution of the protein can be introduced. Asecond reactor 10a is provided so as to make it possible to transmit thegas through either of the reactors by means of a multiple path valve 12.The gas mixture supplied from the dosing apparatus 2 is transmitted tothe reactor via a magnet valve 14 and a relatively narrow tube 15 inwhich a turbulent flow is obtained so as to obtain a homogeneous gasmixture. The reactor 1, the valves 12 and 14, and the tube 15 areencased in a chamber 16 which can be heated, the heat being controlledby a thermostate by means of which the temperature can be varied between30- 100 C.

The dosing apparatus 2 in FIG. 2 comprises a number of containers 21each of which contains one of the reagents to be used. Each container issurrounded by a chamber 32 and temperature of which is held constant bya thermostat. By choosing a suitable temperature a desired vaporpressure can be obtained. The carrier gas is supplied to the container21 via a tube 22 which is extended to the bottom of the container. Thetube 22 is via a regulating valve 23 connected to a valve unit 24comprising a magnet valve 24a connected to each container 21. By meansof this valve unit the carrier gas can be supplied to one or severalcontainers 2]. The carrier gas is supplied to the valve unit 24 from agas container 27 via a tube 26 which comprises a flow-regulating valveso as to keep the flow at the input of the valve unit 24 constant. Eachof the containers 21 are furthermore connected to a valve unit 30,having a design equal to that of the valve unit 24. The valve unit 30and the tube that connects the unit with the containers 21 are encasedin a heat chamber 31 which preferably has a temperature of 100 C. so asto prevent a condensation of the reagent. The gas mixture is supplied tothe reactor via a tube The absorber 3 consists of a tube surrounded by achamber 34 which can be cooled to 70 C. and heated to about 250 C. Theinner wall of the tube is provided with a thin layer of substance inwhich the amino acid derivatives can be sorbed, the layer consisting ofa material which is not vaporized at a temperature of 250 C. The layermight consist of compounds having high molecule weight, e.g.,hydrocarbons or silicon compounds. So called silicon oils are also wellsuited for this purpose. The absorber 3 is connected to the reactor 1via a valve 35 having a rotatable valve body and 8 inlets and outlets.The valve 35 is furthermore connected to a low-vacuum pump 4, via a tubecomprising a three path valve 37 and a cooling trap 38, and ahigh-vacuum pump 5 via a tube comprising a cooling trap 39. Each valve35 is furthermore connected to the analyzer 6 and a container 40containing pure inert gas. The valve 36 is incased in a heat chamber 36which preferablyis kept at a temperature of about 250 C. The valve 35can be adjusted to two positions. In FIG. the channels 4l of the valvebody connect one end of the absorber tube with the reactor 1 and theother end of the absorber tube with the high vacuum pump 5. The valvebody can be turned one eighth of a full turn. In the position thusobtained one channel 41 connects the reactor 1 and the three-path-valve37 while other channels 4] connect the absorber tube 3 with the gascontainer 40 as well as with the analyzer 6.

The operation of the apparatus will now be described briefly. A solutioncomprising a protein to be analyzed is introduced into the reactor 1through the opening 13, and the complete system is flushed with theinert carrier gas. The temperature of the heat chamber 16 is adjusted toabout 50 C. The reagents to be used are disposed in the containers 21,i.e., coupling agent, e.g., isothiocyanate, buffer, blocking agent andsplitting agent as well as hydrolyzing agent. The temperature of theheat chambers of the containers 21 are then adjusted to temperaturesgiving desired partial pressures of the respective reagents. By means ofthe carrier has the reagents are supplied to the reactor 1, in which theprotein is disposed. the reactions A-D described above being performed.Between the different reaction steps the reactor is flushed with theinert carrier gas. During the reaction steps the valves 35 and 37 areadjusted so as to make the gas that leaves the reactor 1 pass theapparatus through the three-path-valve 37.

The reactor does now contain the remaining part of the protein which isrelatively loosely bound to the solid support. These last-mentionedsubstances are the amino acid derivatives, remainders of the usedreagents and substances formed by undesired side reactions. In order toget rid of the reagents and the side products the three-path-valve 37 isnow adjusted so as to connect the reactor 1 to the low vacuum pump 4,the reactor being evacuated to a vacuum of about 10 Torr, thetemperature of the reactor simultaneously being raised to 70-90 C. Thevolatile reagents and the side reaction substances will then be trappedin the cooling trap 38. After a few minutes the valve 35 is adjusted soas to connect the reactor 1 to the absorber 3 which in turn is connectedto the highvacuum pump. The reactor and the absorber are evacuated so asto obtain a vacuum of at least 10 Torr. The decreasing of the pressureimplies that the step E is carried out, i.e., the amino acid derivativeleaves the reactor and passes to the absorber. This reaction isfacilitated by a weak carrier gas flow which passes through the reactorand the absorber. The amount of gas must not exceed the amount that maypass the system through an opening having a diameter of 0.02-0.04 mm.After a few minutes the valve 35 is switched so as to fill the absorber3 with inert gas from thegas container 40, the temperature of thechamber 34 simultaneously being raised 250-300 C. When the absorber hasthe desired temperature a flow of inert carrier gas is made to pass fromthe gas container 40 through absorber to the analyzer 6. If the analyzeris a gas chromatograph the complete sample should be introduced within5-10 seconds. The above-mentioned step F is now carried out and theapparatus is ready for the next step of the sequence analysis.

If the amino acids are identified with a gas chromatograph which worksvery fast, several reactors can be connected to the same gaschromatograph.

The apparatus according to the invention is preferably controlledautomatically. Thus, the valves and the temperatures could be adjustedby a program apparatus known per se. The apparatus could furthermore beused for analyzing amino acids of protein hydrolysates since thereactions in the sequencing procedure are also applicable for free aminoacids.

We claim:

1. Method for determining the sequence of the amino acids in a proteinby making the protein react with a coupling agent which is coupled tothe amino end of the protein molecule, by splitting the obtained proteinderivative at the first peptide binding so as to form an amino acidderivative and a remaining part of the protein, and by identifying theamino acid or the amino acid derivative, the determining furthercomprising subsequent couplings and splittings of the remaining part ofthe protein and identifications of the amino-acid derivatives,characterized in, that the protein is applied to the surface of a solidsupport, that the coupling agent is supplied in gas phase to the appliedprotein and that the amino acid derivative split off is removed in gasphase from the remaining part of the protein applied to the solidsupport.

2. Method according to claim 1, characterized in, that the proteinapplied to the solid support istreated with a blocking means whichblocks reactive groups of the protein molecule, and thus preventsundesired side reactions of the protein and the splitting agent,

3. Method according to claim 1, characterized in, that the protein isadsorbed on the surface of a solid support, consisting of massivegranulates without micropores.

4. Method according to claim 1, characterized in, that the couplingagent and the blocking agent supplied to the protein are mixed with aninert carrier gas.

5. Method according to claim 1, characterized in, that the couplingagent consists of a cyanate.

6. Method according to claim 1, characterized in, that the couplingagent consists of an isothiocyanate.

7. Method according to claim 1, characterized in, that the blockingagent consists of a silicon containing compound.

8. Method according to claim 1, characterized in, that after thesplitting the adsorbed amino acid derivative is made 'to react with avapor pressure increasing agent in order to facilitate the separation ofthe derivative from the remaining part of the protein.

9. Method according to claim 8, characterized in, that the vaporpressure increasing agent consists of a silicon-containing compound.

10. Method according to claim 9, characterized in, that the siliconcontaining groups are removed from the remaining part of the protein bya hydrolysis of vaporized water when the volatile amino acid derivativehas been removed from the remaining part of the protein adsorbed on thesolid support 11. Method according to claim 3', characterized in, thatthe amino acid derivative is removed from the remaining part of theprotein adsorbed on the solid support by raising the temperature.

12. Method according to claim 3, characterized in, thatthe amino acidderivative is removed from the remaining part of the protein adsorbed onthe solid support by lowering the pressure.

13. Method according to claim 3, characterized in, that the amino acidderivative is removed from the remaining part of the protein adsorbed onthe solid support by raising the temperature and lowering the pressure.

14. Method according to claim 1, characterized in, that the removal ofthe amino acid derivative is preceded by a removal of undesired sideproducts.

15. Method according to claim 1, characterized in, that the amino acidderivative is transferred to a cooled absorber and by heating of theabsorber transferred to an analyzer.

16. Apparatus for determining the sequence of the amino acids in aprotein characterized in, that the apparatus comprises a reactor,-including a solid support on which the protein is adsorbed, means forgenerating a flow of inert carrier gas, means for supplying this gasthrough the reactor, means for mixing the gas'with a coupling agent ingas phase and for mixing the carrier gas with other reagents in gasphase, e.g., a blocking agent, the apparatus further comprising anabsorber inwhich the volatile amino acid derivatives formed in thereactor are absorbed, means for heating the reactor in order tofacilitate a transferring of the amino acid derivative from the reactorto the absorber, an analyzer for identifying the amino acid comprised inthe amino acid derivative and, means for heating the absorber so as tofacilitate thetransferring of the amino acid derivatives from theabsorber to the analyzer.

2. Method according to claim 1, characterized in, that the proteinapplied to the solid support is treated with a blocking means whichblocks reactive groups of the protein molecule, and thus preventsundesired side reactions of the protein and the splitting agent. 3.Method according to claim 1, characterized in, that the protein isadsorbed on the surface of a solid support, consisting of massivegranulates without micropores.
 4. Method according to claim 1,characterized in, that the coupling agent and the blocking agentsupplied to the protein are mixed with an inert carrier gas.
 5. Methodaccording to claim 1, characterized in, that the coupling agent consistsof a cyanate.
 6. Method according to claim 1, characterized in, that thecoupling agent consists of an isothiocyanate.
 7. Method according toclaim 1, characterized in, that the blocking agent consists of a siliconcontaining compound.
 8. Method according to claim 1, characterized in,that after the splitting the adsorbed amino acid derivative is made toreact with a vapor pressure increasing agent in order to facilitate theseparation of the derivative from the remaining part of the protein. 9.Method according to claim 8, characterized in, that the vapor pressureincreasing agent consists of a silicon-containing compound.
 10. Methodaccording to claim 9, characterized in, that the silicon containinggroups are removed from the remaining part of the protein by ahydrolysis of vaporized water when the volatile amino acid derivativehas been removed from the remaining part of the protein adsorbed on thesolid support.
 11. Method according to claim 3, characterized in, thatthe amino acid derivative is removed from the remaining part of theprotein adsorbed on the solid support by raising the temperature. 12.Method according to claim 3, characterized in, that the amino acidderivative is removed from the remaining part of the protein adsorbed onthe solid support by lowering the pressure.
 13. Method according toclaim 3, characterized in, that the amino acid derivative is removedfrom the remaining part of the protein adsorbed on the solid support byraising the temperature and lowering the pressure.
 14. Method accordingto claim 1, characterized in, that the removal of the amino acidderivative is preceded by a removal of undesired side products. 15.Method according to claim 1, characterized in, that the amino acidderivative is transferred to a cooled absorber and by heating of theabsorber transferred to an analyzer.
 16. Apparatus for determining thesequence of the amino acids in a protein characterized in, that theapparatus comprises a reactor, including a solid support on which theprotein is adsorbed, means for generating a flow of inert carrier gas,means for supplying this gas through the reactor, means for mixing thegas with a coupling agent in gas phase and for mixing the carrier gaswith other reagents in gas phase, e.g., a blocking agent, the apparatusfurther comprising an absorber in which the volatile amino acidderivatives formed in the reactor are absorbed, means for heating thereactor in order to facilitate a transferring oF the amino acidderivative from the reactor to the absorber, an analyzer for identifyingthe amino acid comprised in the amino acid derivative and, means forheating the absorber so as to facilitate the transferring of the aminoacid derivatives from the absorber to the analyzer.